Elucidating the effects of amide side chain interactions in flexible biomolecules

V. Alvin Shubert, Esteban E. Baquero, Jasper R. Clarkson, Timothy S. ZwierDepartment of Chemistry, Purdue University

West Lafayette, IN 47907

M. J. TubergenDepartment of Chemistry, Kent State University

IntroductionPrevious studies on the single conformation IR and UV spectroscopy and dynamics of conformational isomerization.

Tryptamine Melatonin N-acetyl tryptophan methyl

amide NATMAAbove studies have led us to custom build molecules with different types of flexibility built in.

p-methoxyphenethylacetamide 2-phenoxyethylacetamide O-(acetamidoethyl)-N-acetyltyramine

MPEAmide POEA OANAT

O N

O

N

O

O N

O

ON

O

RI03

59th MSS, WG02

Introduction

Doubly substituted molecules

• Two branches to potential energy surface (PES)– Can branches be decoupled?

– What types of chains allow for interchain interactions (e.g. H-bonding)?

– What types of chains hinder interchain interactions?

• Dynamics studies– Breaking H-bonds

– Put energy into one chain, observe effects on other chain

N-(2-phenylethyl)-acetamide

N,N’-(1,4-phenylenedi-2,1-ethanediyl)bis acetamide

1,4-phenylenebis(oxy)bis-N-methylpropanamide-

2,2’-[1,4-phenylenebis(oxy)]bis-ethanamide

3,3’-(1,4-phenylenedi)bis-(N-methylpropanamide)

O-(acetamidoethyl)-N-acetyltyramine

1,4 (N-methylpropanamide-N’-ethylacetamide)benzene

N-methyl-benzenepropanamide

3-phenoxy-N-methylpropanamide

2-phenoxyethylacetamide

NH

O

R

NH

R

O

NH

OR

O

NH

O

OR

Molecules under study

What types of low energy conformations will be formed for double chain molecules?

• How does the presence of the second chain affect the conformational preferences of the first chain and vice versa?

• Given n low energy structures for one chain and m for the other, might expect nXm conformations.

• However, this assumes two things:– Chains do not asymmetrize the benzene ring– Chains are non interacting

• Since chains do asymmetrize the benzene ring,# of conformations expected = nXmX # of spectroscopically distinct orientations

• If chains do interact, this may lead to structures containing individual chain conformations that were high energy in single chain molecules.

The number of possible conformations gets very large very fast.

Disconnectivity Diagrams

-44.0

-40.0

-36.0

-32.0

-28.0

-24.0

-20.0

-16.0

Energy (kcal/mol)

ON

OO N

O

O N

O

N

O

POEA

OANAT

MPEAmide

-415.0

-410.0

-405.0

-400.0

-395.0

-390.0

-385.0

-380.0

-375.0

-370.0

-365.0

-360.0

-355.0

-350.0

-89.0

-85.0

-81.0

-77.0

-73.0

-69.0

-65.0

-61.0

OPTIM.2.3 and Disconnect, David J. Wales, Cambridge University

Experimental methodsResonant 2 photon ionization (R2PI): Records spectra in mass selective fashion

Biomolecule* (S1)

Biomolecule (S0)

Biomolecule+ + e-

Hol

e-bu

rn

Pro

be

Conformer A Conformer B

Hol

e-bu

rn

Pro

be

UV Source fixed: Provides selectivity IR Source tuned

R2PI: Electronic SpectrumResonant ion dip infrared spec-troscopy (RIDIRS): Conforma-

tion specific IR spectrum

UV-UV Hole-burning: Conformation specific electronic spectrum

Computational methodsSearch for lowest energy structures

1. Draw molecule in MacroModel, use conformation search.

2. Sift through structures found by MacroModel (Typically > 500).

3. Use unique structures as starting structures for optimizations using Gaussian03. Optimize at B3LYP/6-311+G* level of theory.

4. Using Gaussian03 and optimized structures, calculate infrared frequencies and intensities.

800

600

400

200

0

Infr

are

d Inte

nsi

ty (

KM

/mole

)

3500300025002000150010005000Frequency (cm

-1)

R2PI and UV-UV hole-burning spectra of OANAT

• Six conformations resolved to date.

• Four conformations (C,D,E,F) have origins within 85 cm-1 of 35,630 cm-1.

• Two conformations (A,B) are red shifted by more than 1000 cm-

1 from the other four.

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

Ion

inte

nsity

36400362003600035800356003540035200350003480034600photon energy (cm

-1)

HB at 34,536.4 cm-1

(also origin)

HB at 34,539.3 cm-1

(also origin)

HB at 35,549.2 cm-1

(also origin)

HB at 35,607.5 cm-1

(also origin)

HB at 35,631.6 cm-1

(origin at 35,622.05 cm-1

)

HB at 35,711.1 cm-1

(also origin)

R2PI of OANAT

A

BC

D

E

F

RIDIR spectra in amide NH stretch region

• RIDIR spectra indicate three classes of conformations for OANAT.

• Two stretches seen, one from each chainIo

n in

tens

ity (

arbi

trar

y un

its)

352035003480346034403420340033803360

photon energy (cm-1

)

A

B

C

D

C

B

A

A

B

CD

E

F

OANATPOEAMPEAmide

MPEAmide

POEA

ON

O

O N

O

O N

O

N

O

OANAT

Summary of Experimental Results

• 2 major conformations for alkyl chain

• 1 major conformation for alkoxy chain

• Expected 8 conformations in OANAT, only found 6

• Two classes of conformations:– H-bonding between chains

– Independent chains• Only 3 independent chain conformations found, not 8 as expected

Interacting chains Independent chains

Computational resultsSingle chain molecules

NPEA, NMBPA, PNMP, and POEA• Generally, find few low energy structures (< 0.2 kcal/mol, relative)

(3, 2, 1, and 1, respectively)• Most significant differences seen in vibrational frequencies are those associated with the ether

linkages of PNMP and POEA

MPEAmide, NPEBA, NMPNEA, PBNMP, PBOBEA, and OANAT• Generally, find many low energy structures (6-10)• Many have non-interacting chains and can be additively constructed from conformations found for single chain

molecules• Little shift seen in vibrational frequencies compared to single chain molecules when chains are non-interacting• If chains interact (i.e. form an interchain H-bond), shifts are seen in frequencies, especially for NH and CO stretches

Double chain molecules

OCH3

Combining single chains onto one molecule: Structures

NPEA

0.000 kcal/mol

0.172 kcal/mol

NMBPA

0.000 kcal/mol

+

NMPNEA

0.283 kcal/mol

0.329 kcal/mol

0.240 kcal/mol

0.414 kcal/mol

NH

O

NH

O

NH

NH O

O

Computational resultsCombining single chains onto one molecule: Vibrational frequencies

2000

1500

1000

500

0

Infr

are

d I

nte

nsi

ty (

KM

/mo

le)

3600320028002400200016001200

Frequency (cm-1

)

NPEA (0.000 kcal/mol) + NMBPA (0.000 kcal/mol)

NPEA (0.172 kcal/mol) + NMBPA (0.000 kcal/mol)

NMPNEA (0.283kcal/mol)

NMPNEA (0.240 kcal/mol)

NMPNEA (0.329 kcal/mol)

NMPNEA (0.414 kcal/mol)NH

O

NPEA

NH

O

NMBPA

NH

NH O

O

NMPNEA

-6

-4

-2

0

2

4

6

35003480346034403420340033803360

-1.0

-0.5

0.0

0.5

ion

inte

nsi

ty (

arb

itra

ry u

nits

)

35203510350034903480

photon energy (cm-1

)

What if chains do interact? Example, OANAT

Interacting chains

Independent chains

A

B

C

D

E

F

B3LYP/6-31+G*, 6-311+G*Gaussian98 and Gaussian03

Alkyl NH · · · Alkoxy CO Alkyl CO · · · Alkoxy NH

Alkyl NH · · · Alkoxy CO

Alkyl CO · · · Alkoxy NH

NPEBA: H-bond conformation at 0.006 kcal/mol, 3 more with H-bonds at < 0.75 kcal/mol

PBNMP: Weak H-bonding in 2 conformations > 1.4 kcal/mol

PBOBEA: No conformations with H-bonds found

NMPNEA: Lowest energy conformation contains H-bond between NH of CONH and CO of NHCO

Discussion

• What types of chains lead to interactions?– NHCO ordering leads to more interaction than CONH– Alkyl chains allow interaction between chains– O connecting chain to ring hinder interaction between chains– Different orderings of amide groups on opposite chains allows for more interaction

• How does interaction effect number of conformations?– Interchain interaction tends to increase number of low energy conformations

• Best design for a molecule with two decoupled branches to PES is to use CONH ordering for amide group and use O to connect chain to ring

NH

NH

O O

NH

NH

OO

NH

NH

O

O

O NH

ONH

O O

Summary

• Six conformers of OANAT have been resolved to date• Doubly substituted molecules are more than sum of their parts – simply building

structures based on low energy conformations of single arm molecules is not adequate. Interactions between chains act to lower the energy of some chain configurations.

• Calculations are only a rough guide at our level of theory.

Future Work

• Spectra in the 1000-2000 cm-1 region• SEP and SEP hole-filling• IR hole-filling: Selective excitation of amide in each chain• Complete search for low energy conformations, disconnectivity diagrams• Build different types of chains (i.e. vary number of carbons between amide group and

chromophore, use different chromophores, and/or substitute into different positions, ortho or meta)

AcknowledgementsPeople

Prof. Timothy S. Zwier

The Zwier Group

Jasper R. Clarkson

Esteban Baquero

Tracy LeGreve

Bill James

Jaime Stearns

Talitha Selby

Josh Newby

Prof. Michael J. Tubergen

Prof. David Wales

Dr. Dave Evans

Prof. Mark Lipton

Kevin Worrel

Funding

National Science Foundation